Cloning and expression of the rop18 from Toxoplasma gondii in plasmid vectors

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects mammals and birds, including humans. The ROP18 protein was described as an important virulence factor of the parasite, which may make it a potential candidate for protection against this parasite. The present study aimed to clone, sequence, and express the ROP18 gene in plasmid vectors. The partial fragment of rop18 was amplified by PCR, and the DNA was cloned into vectors pcDNA 3.1-TOPO and pTrcHis-TOPO. Subsequently, they were transformed into Escherichia coli TOP10 cells. The confirmation of the reading frame of the gene in the plasmids was performed by sequencing. The obtained vectors pcDNA-rop18+were transfected into VERO cells, which were fixed on slides and submitted to the technique of indirect fluorescent antibody test (IFAT) with anti-histidine, and pTrcHis-rop18+ were transformed in E. coli BL21 to evaluate the expression of the protein. After expression in E. coli BL21, the recombinant rROP18 proteins were purified on a Ni-NTA column under denaturing conditions and visualized on an SDS polyacrylamide gel (42 kDa) with a concentration of 230 µg/ml. A reaction with anti-histidine was observed in Vero cells transfected with pcDNA-rop18+. In conclusion, the rop18 partial gene was successfully cloned and expressed in Vero cells and in E. coli, producing a protein under insoluble conditions, and the characteristics of the hydrophilic antigen suggest its use for studies of vaccine development of recombinant proteins or DNA and tests for diagnosis.